Sex differences in cachexia and branched‐chain amino acid metabolism following chemotherapy in mice

Abstract Chemotherapy is a major contributor to cachexia, but studies often investigate male animals. Here, we investigated whether sex modifies the effects of chemotherapy on cachexia and BCAA metabolism. Ten‐week‐old CD2F1 male and female mice were treated with the chemotherapy drug cocktail folfiri (50 mg/kg 5‐fluorouracil, 90 mg/kg leucovorin, and 24 mg/kg CPT11) (drug) or vehicle twice a week for 6 weeks. Insulin tolerance tests were conducted and BCAA levels and metabolism were measured in plasma and tissues. Drug treatment reduced body and skeletal muscle weights and anabolic signaling in both sexes, with females showing worsened outcomes (p < 0.05 for all). Drug treatment increased plasma BCAA only in males, but BCAA concentrations in the skeletal muscle of both sexes were decreased; this decrease was more profound in males (p = 0.0097). In addition, muscle expression of the BCAA transporter LAT1 was reduced; this reduction was more severe in females (p = 0.0264). In both sexes, the (inhibitory) phosphorylation of BCKD‐E1αser293 was increased along with decreased BCKD activity. In the liver, drug treatment increased BCAA concentrations and LAT1 expression, but BCKD activity was suppressed in both sexes (p < 0.05 for all). Our results demonstrate that altered BCAA metabolism may contribute to chemotherapy‐induced cachexia in a sex‐dependent manner.


| INTRODUCTION
Sex is a major risk factor for cancers (Kim et al., 2018).Affecting 20%-80% of cancer patients dependent on cancer stage and type is cachexia, a devastating body and skeletal muscle wasting syndrome (Lim et al., 2020).Systemic inflammation, insulin resistance, increases in energy expenditure, negative protein/energy balance, appetite loss, anorexia, and fatigue are all experienced in cachexia (Evans et al., 2008).Chemotherapy is also a major contributor to skeletal muscle loss and cachexia (Ballarò et al., 2019;Barreto, Waning, et al., 2016;Pin et al., 2019).
Many of the previous studies on chemotherapyinduced cachexia are conducted in male animals (Barreto, Mandili, et al., 2016;Barreto, Waning, et al., 2016;Pin et al., 2019;Rosa-Caldwell & Greene, 2019).However, there is evidence for sex differences in BCAA metabolism.Compared to females, males have higher concentrations of the BCAA (Costanzo et al., 2022) and greater leucine oxidation after endurance exercise (Mittendorfer et al., 2002).Further, estrogen decreases BCKD activity, but this study only investigated female animals (Obayashi et al., 2004).Here, we hypothesized that chemotherapy would induce greater catabolism of BCAA in tissues of male mice, and that this change would be associated with more severe cachexia.

| Animals
Fourteen male and 14 female 8-week-old CD2F1 were purchased from Charles River and Envigo Laboratories.Because of supply issues from our original supplier, female mice of the same strain and at similar age were obtained from Envigo.Mice were acclimatized and housed in the vivarium with free access to food (Purina 5015*, LabDiet, St. Louis, MO) and water.All animals were aged until 10 weeks before treatment.For females, estrous tracking occurred 1-week prior to treatment to ensure that female mice were in the same stage of their estrous cycle.Chemotherapy treatment began when female mice were in di-estrous.Mice were separated into four groups: male vehicle, male drug, female vehicle, female drug (n = 7 for all groups).Mice were administered intraperitoneally (i.p.) either the chemotherapy drug cocktail folfiri (50 mg/ kg 5FU (#F6627), 90 mg/kg Leucovorin (#F7878) and 24 mg/kg CPT11 (#I1406)) (drug) or vehicle ((3.8% DMSO (#D5879), all from Sigma Aldrich, St. Louis, MO) in saline) twice a week for 6 weeks.The two weekly doses were separated by 2 days.This drug cocktail is typically used in the treatment of colorectal cancer (Tabernero et al., 2015).Drug dosages were taken from a previous study whereby this cocktail induced atrophy in the skeletal muscle of mice (Barreto, Mandili, et al., 2016).Bodyweight and food consumption were recorded daily.However, to account for minor differences in baseline values, body weight was presented as weekly variations relative to Day 0 as presented by Barreto et al. (2016).At the end of the study, animals were euthanized via cervical dislocation at least 24 h after the last chemotherapy dose.Following sacrifice, the gastrocnemius, tibialis anterior and quadriceps were collected.Visceral white adipose tissue, kidney, liver, and spleen were also collected.Tissues were weighed, flash-frozen in liquid nitrogen and stored at −80°C until analysis.The gastrocnemius muscle was used for the subsequent analyses, as a sex difference was observed in the weight of this muscle following treatment.

| Insulin tolerance test
At both the third and sixth weeks of treatment, insulin tolerance tests (ITT) were performed.With at least 24 h after their previous chemotherapy dose, mice were fasted for 6 h and 0.75 units/kg of insulin (Eli Lilly, Humulin R, #00586714, Indianapolis, IN) was administered via subcutaneous injection.Blood samples were collected from the saphenous vein on glucose strips (Alpha TRAK, #71681, Parsippany, NJ) at 0 (baseline), 5, 15, 30, and 120 min post insulin injection.The strips were inserted into a glucometer (Alpha TRAK, #71675-01) to obtain glucose concentrations.

| Protein synthesis (SUnSET analysis)
With at least 24 h after their last chemotherapy dose, mice were starved for 3 h in order to limit the effects of nutrient uptake on protein synthesis.Thirty minutes prior to euthanasia, mice were intraperitoneally injected with 0.040 μmol/g bodyweight of puromycin (Sigma Aldrich, #P8833) in saline.Skeletal muscles were removed after euthanasia and muscle proteins were immunoblotted against an anti-puromycin antibody and corrected to their Ponceau S staining.

| Statistics
All immunoblot analyses were quantified and adjusted to their corresponding γtubulin values.Graphs were drawn using Prism 10 (GraphPad software).Because male and female mice were obtained from separate vendors, we used unpaired t-tests to measure treatment differences within each sex.We calculated percentage changes due to treatment (within each sex) and then used an unpaired t-test to compare whether those treatment-induced percentage changes differed between the sexes.For the ITT data, within sex, a twoway-ANOVA was used to measure blood glucose differences following chemotherapy across all time points, followed by area under the curve (AUC) analysis.We also measured and compared the slopes of the lines between the 5 and 15 min time points in drug-treated male and female mice at 6 weeks.Results were expressed as mean ± standard deviation (SD).Significance was determined as p < 0.05.

| Drug-treated female mice have worsened outcomes for body and skeletal muscle weight following chemotherapy
Following 6 weeks of chemotherapy treatment, drugtreated males lost ~10% body weight, while drug-treated females lost ~15% (relative to their baseline weight, Figure 1a; raw body and tissue weights are shown in Table S2).At Week 6, body weight loss was higher in female than in male (Figure 1b).Drug treatment did not affect food intake (Figure 1c,d).The loss of body weight in drug-treated animals was consistent with significant decreases in the F I G U R E 1 Drug-treated female mice have worsened outcomes for body and skeletal muscle weight following chemotherapy.Male and female 10-12-week-old CD2F1 mice were treated with either vehicle (V, 3.8% DMSO in saline, black bars) or a chemotherapy drug cocktail (D, Drug: 50 mg/kg 5-FLU, 90 mg/kg Leucovorin, 24 mg/kg CPT11, white bars) twice a week for 6 weeks.Body weight (a, b) and food intake (c, d) were recorded daily for 6 weeks.Animals were euthanized at least 24 h after their last chemotherapy dose.Weights of the skeletal muscles, corrected for final body weight (FBW): gastrocnemius (e), tibialis anterior (f) and quadriceps (g).Data are mean ± SD; n = 7 animals per group.
F I G U R E 2 Chemotherapy causes greater insulin resistance in males, but drug-treated female mice experience worsened outcomes for anabolic and catabolic signaling.Animals were treated as described in Figure 1.(a) Insulin binds to the insulin receptor (IR) leading to the phosphorylation of IRS-1 on tyrosine residues and the eventual activation of AKT.AKT activation leads to an increase in insulin stimulated glucose uptake in skeletal muscle.AKT also indirectly activates mTORC1, leading to the phosphorylation of S6K1 and 4E-BP1, ultimately leading to an increase in protein synthesis.The BCAA, transported into skeletal muscle through LAT1, activate components in the sestrins/ gator/RAG/ragulator pathway, leading to the activation of mTORC1.During Weeks 3 and 6 of treatment, with at least 24 h after their previous chemotherapy dose, male (b, e) and female (c, f) mice were starved for 6 h and then underwent an ITT.Blood glucose AUC in both sexes is shown (d, g).Immunoblotting and quantified data for p-AKT ser473 (h, i), p-S6K1 thr389 (h, j), p-S6 ser235/236 (h, k) and p-4E-BP1 thr37/46 (h, l) in the gastrocnemius muscle are shown.Immunoblots are shown for total AKT, S6, S6K1 and 4E-BP1 in the gastrocnemius (m).Protein synthesis was measured via the SUnSET analysis at least 24 h from their previous chemotherapy dose.Thirty minutes prior to euthanasia, mice were injected with 0.040 μmol/g bodyweight of puromycin.Following euthanasia, tissues were isolated.Skeletal muscle proteins were immunoblotted against an anti-puromycin antibody and corrected to their respective Ponceau S staining (n, o).Immunoblotting and quantified data for p-FoxO3a ser253 (p, q), MuRF1 (p, r), and ubiquitinated proteins (s, t) in the gastrocnemius muscle are shown.Data are mean ± SD; n = 5-7 animals per group.
weights of the gastrocnemius (Figure 1e), tibialis anterior (Figure 1f) and quadriceps (Figure 1g) muscle.The loss of gastrocnemius muscle weight in females was more severe compared to males (Figure 1e).There were no effects of drug treatment on adipose tissue and kidney weights, but both sexes showed a significant increase in spleen weight, while only drug-treated females showed an increase in liver weight compared to controls (Figure S1A-D).Because of the sex difference in response to chemotherapy in the gastrocnemius muscle weight, all the subsequent musclebased measures were done in this muscle.Protein expression of gastrocnemius muscle contractile proteins MHC-1, troponin, and tropomyosin were decreased in both sexes following chemotherapy (Figure S1E-H).

| Chemotherapy causes greater insulin resistance in males, but drug-treated female mice experience worsened outcomes for anabolic and catabolic signaling
A simplified diagram of insulin, BCAA and mTORC1 signaling is shown (Figure 2a).At Weeks 3 and 6 of treatment, male (Figure 2b,e), but not female (Figure 2c,f) drug-treated mice showed impaired insulin tolerance, corresponding with higher blood glucose AUC (Figure 2d,g).Because the observed sex differences during the ITT could be due to higher basal (pre-insulin injection) glucose levels in drug-treated animals, we also calculated the slopes of the glucose curves between time points 5 and 15 min and found no differences at 6 weeks of chemotherapy in either sex (mean slope for male: −0.3217, mean slope for female: −0.3386, p = 0.7052).
In the gastrocnemius muscle, there was a decrease in the phosphorylation of AKT ser473 , S6K1 thr389 , S6 ser235/236 and 4E-BP1 thr37/46 compared to controls (Figure 2h,i).The decrease in phosphorylation of S6K1 thr389 in drug-treated females was more severe compared to males (Figure 2h,j).Total proteins of these phosphorylated targets were not affected by treatment (Figure 2m).Protein synthesis measured by SUnSET analysis was also decreased following drug treatment in both sexes (Figure 2n,o).For catabolic signaling, only drug-treated female mice showed decreases in p-FoxO3a ser253 (Figure P, Q), consistent with a treatmentinduced increase in MuRF1 expression (Figure 2p,r) and ubiquitinated proteins in females (Figure 2s,t).

| Following chemotherapy, the reduction in skeletal muscle BCAA concentrations is more severe in males
Concentrations of leucine, isoleucine, valine and total BCAA were reduced in drug-treated animals (Figure 3a-d).These decreases were more severe in males.Only drug-treated males showed a decrease in arginine concentrations (Figure 3e), while minimal differences were found for concentrations of alanine, phenylalanine, serine and glutamate (Figure 3f-i).Both sexes showed decreases in the expression of AA transporters SNAT1 (Figure 3j,k) and LAT1 (Figure 3j,l) following chemotherapy; however, the decrease in the expression of LAT1 was more severe in females than in males (Figure 3j,l).

| Skeletal muscle BCAA catabolism is decreased following chemotherapy in both sexes
A simplified diagram of the first two steps in BCAA catabolism is shown (Figure S2).Protein expression of BCAT2 was unchanged following drug treatment (Figure 4a,b).Expression of BCKD-E1α was unchanged in males, but decreased in drug-treated female animals (Figure 4a,c).BDK was unchanged following drug treatment (Figure 4a,d).However, the inhibitory phosphorylation of BCKD-E1α ser293 was increased in both sexes (Figure 4a,e), corresponding with decreased activity of this dehydrogenase complex (Figure 4f).

| Chemotherapy treatment leads to elevated concentrations of liver BCAA in both sexes, but only males show elevated plasma BCAA
Only drug-treated males showed elevated plasma concentrations of leucine, valine (but not isoleucine) and total BCAA (Figure 5a-d).We also found elevated concentrations of leucine, valine (but not isoleucine) and total BCAA (Figure 5e-h) in the livers of drug-treated mice from both sexes, with males showing a greater increase in valine (Figure 5g).LAT1 expression was increased in the liver from both sexes (Figure 5i,j).There were no significant treatment effects on the protein expression of BCKD-E1α and BDK (Figure 5i,k,l).However, similar to skeletal F I G U R E 3 Following chemotherapy, the reduction in skeletal muscle BCAA concentrations is more severe in males.Animals were treated as described in Figure 1.Concentrations of leucine (a), isoleucine (b), valine (c), total BCAA (d), arginine (e), alanine (f), phenylalanine (g), serine (h) and glutamate (i) in the gastrocnemius muscle were measured by HPLC.Immunoblotting and quantified data for SNAT1 (j, k) and LAT1 (j, i) in the gastrocnemius muscle.Data are mean ± SD; n = 6-7 animals per group.muscle, the inhibitory phosphorylation of BCKD-E1α ser293 was higher following drug treatment (Figure 5i,m), corresponding with decreased activity of BCKD in the liver (Figure 5n).

| The BCAA are positively correlated with gastrocnemius muscle weight, LAT1 transporter expression and BCKD activity
Total BCAA showed a positive correlation with gastrocnemius muscle weight (Figure 6a), protein expression of LAT1 (Figure 6b), and BCKD activity (Figure 6c).Positive correlations were also observed between muscle weight and LAT1 (Figure 6d) and BCKD activity (Figure 6e).

| DISCUSSION
While previous studies have examined the effect of cancer and/or chemotherapy on muscle mass and protein turnover (Ballarò et al., 2019;Barreto, Mandili, et al., 2016;Barreto, Waning, et al., 2016;Mora & Adegoke, 2021;Pin et al., 2019), we report, to our knowledge for the first time, sex differences in circulating and tissue BCAA availability and muscle BCAA transporter expression following administration of antineoplastic drugs to mice (Table 1).We show that body and gastrocnemius muscle weight losses in response to chemotherapy were more severe in female compared to male, consistent with greater loss of mTORC1/S6K1 signaling in muscles of female.However, drug-treated male mice developed impaired insulin tolerance and experienced more severe decreases in skeletal muscle BCAA concentrations.The observed sex differences in BCAA levels might not be due to changes in LAT1, an established BCAA transporter, because treatmentinduced suppression of LAT1 expression was surprisingly more pronounced in females.Collectively, these data reveal previously undocumented sex-and tissue-specific alterations to muscle BCAA metabolism in response to chemotherapy and suggest that these changes may predict severity of chemotherapy-related cachexia.
Identifying interventions that can prevent/mitigate cachexia has remained a holy grail in the field.For example, while the muscle anabolic effects of BCAA are incontrovertible, administration of these AAs has yielded minimal benefit on cachexia in humans (Harima et al., 2010;Pimentel et al., 2021;Poon et al., 2004;Soares et al., 2020).The anti-anabolic and pro-catabolic milieu that prevails in cachectic patients and rodents, as indicated by elevated circulating levels of catabolic factors such as tumor necrosis factor, IL-6, and interferon gamma (de Matos-Neto et al., 2015), are likely contributory to the recalcitrance to nutrients in cachectic conditions.Our data from cultured myotubes (Mora & Adegoke, 2024) and the in-vivo data presented here clearly demonstrate that the blunted F I G U R E 4 Skeletal muscle BCAA catabolism is decreased following chemotherapy in both sexes.Animals were treated as described in Figure 1.Immunoblotting (a) and quantified data for BCAT2 (b), BCKD-E1α (c), BDK (d) and p-BCKD-E1α ser293 (e) in the gastrocnemius muscle.BCKD activity was measured from the release of 14 CO 2 from 14 C labeled valine (f).Data are mean ± SD; n = 5-7 animals per group.
response to nutrients might be due to limitations in substrate delivery to muscle and/or accelerated efflux from skeletal muscle to plasma.
One of the main findings of this work is the sex differences in some of the commonly measured indicators of cachexia (body and tissue weights).In this study, drug-treated female mice lost more body and gastrocnemius muscle weights compared to males.This is an important finding, given that skeletal muscle mass is a determinant of chemotherapy dose (Ali et al., 2016) and treatment effectiveness.In healthy subjects, males have greater muscle mass than females (Janssen et al., 2000) F I G U R E 5 Chemotherapy treatment leads to elevated concentrations of liver BCAA in both sexes, but only males show elevated plasma BCAA.Animals were treated as described in Figure 1.Plasma leucine (a), isoleucine (b), valine (c) and total BCAA (d), as well as liver leucine (e), isoleucine (f), valine (g) and total BCAA (h) were measured by HPLC.Immunoblotting and quantified data of LAT1 (i, j), BCKD-E1α (i, k), BDK (i, l) and p-BCKD-E1α ser293 (i, m) in the liver.BCKD activity in the liver (n).Data are mean ± SD; n = 5-7 animals per group.and higher levels of testosterone, the anabolic hormone that enhances muscle mass (Bhasin et al., 2001).In addition, females have lower blood elimination (Kim et al., 2018) and drug clearance (Wagner et al., 2019) for chemotherapy agents, supporting a more catabolic environment in females.Therefore, females may experience more side effects and decreased chemotherapy effectiveness compared to males.Since chemotherapy can also reduce fat mass (Ebadi et al., 2017), we were surprised to see no significant effect of chemotherapy on the adipose tissue weight in both sexes, especially since females have a greater fat mass compared to males (Karastergiou et al., 2012).However, it is important to note that cachexia can occur with or without the loss of adipose tissue (Ni & Zhang, 2020) and since these animals are relatively young in age, it is possible they do not have large fat storage, compared to the fat storage seen in aging (Ponti et al., 2020).
The differences observed between the sexes may be related to sex-dependent alterations in insulin sensitivity.In myotubes, we have shown that chemotherapy leads to decreased insulin-stimulated glucose uptake (Mora & Adegoke, 2021).Our finding of worsened insulin sensitivity only in males is likely related to hormonal differences between the sexes, as estrogen, a predominately female hormone, has protective effects against insulin resistance (De Paoli et al., 2021).Therefore, repeating these experiments in aged post-menopausal female animals may produce different results.
The observed sex-differences in S6K1 phosphorylation are consistent with the reduced muscle weight observed in females.In healthy subjects, minimal sex differences exist for mTORC1 signaling and protein synthesis (Dreyer et al., 2010;West et al., 2012).However, we found that in healthy mice administered chemotherapy, the reduction in mTORC1 signaling was more severe in drug-treated female animals compared to males.Also, drug-treated female mice had increased MuRF1 protein expression, a finding not seen in males.This is contrary to previous literature, whereby males have greater ubiquitin proteasome activity compared to females (Ogawa et al., 2017).However, we only measured protein expression of an E3 ligase, but not proteasome activity per se.Our findings indicate that in healthy mice, administration of chemotherapy led to sex differences in mechanisms related to protein synthesis/breakdown, which may contribute at least in part to the more severe loss of gastrocnemius muscle weight in drug-treated female mice.
Another main finding of this work is the profound sex differences in tissue and circulating BCAA levels.All 20 AAs are required for protein synthesis in skeletal muscle (Wolfe, 2017).Males have higher muscle concentrations of the BCAA (Costanzo et al., 2022).Therefore, higher BCAA concentrations may present a protective effect against chemotherapy-induced cachexia in males, an observation that may partially explain the more severe loss of gastrocnemius muscle weight in females.More severe reductions in muscle BCAA concentrations in males could simply be due to the greater BCAA pools found in males (Costanzo et al., 2022), giving these animals more to lose.However, during skeletal muscle substrate deficits, males tend to oxidize more AAs for energy, while females tend to oxidize more fats (Lamont et al., 2001).Therefore, following chemotherapy, males may generate more energy from muscle AAs to support protein synthesis and abrogate some of the loss of muscle mass from chemotherapy, an effect that is further supported by reduced arginine concentrations seen only in males.Decreased concentrations of BCAA in both sexes is associated with decreased expression of their transporter LAT1, an effect that is more severe in females.The greater loss of LAT1 in females may be explained by the fact that LAT1 can function as a bi-directional transporter (Kahlhofer & Teis, 2022).Female animals lost more muscle, had greater S6K1 suppression, higher MuRF1 expression and greater ubiquitination of proteins, all consistent with a more catabolic environment in females.Therefore, the greater reduction in LAT1 in females may represent an adaptation to limit the loss of BCAA and other AAs in females, thus limiting atrophy.a Indicates significant (p < 0.05) decrease (red) or increase (green) in sex (i.e., male vehicle vs. male drug, or female vehicle vs. female drug).
b Indicates significant delta difference between sexes (i.e., male drug vs. female drug).
Decreased BCAA concentrations in the skeletal muscle following chemotherapy may also be related to altered metabolism of these AAs.The key enzymes involved in BCAA catabolism, mainly BCAT1/2 and BCKD, are elevated in tumors of the liver (Ericksen et al., 2019) and breast (Zhang & Han, 2017).However, these enzymes are not typically studied in the skeletal muscle of cancerinoculated mice, nor following chemotherapy.It is possible that the decreased BCKD activity in the skeletal muscle of both sexes following chemotherapy is a compensatory mechanism to preserve the BCAA pool.
Lastly, it is interesting to note tissue differences in BCAA handling during chemotherapy.The increase in plasma BCAA in males can be related to the release of these AAs from skeletal muscle, a tissue that may rely on the oxidation of AAs during substrate deficits (Lamont et al., 2001).Also, a previous review has outlined that dysregulation of BCAA metabolism in the gut microbiome can result in elevated plasma BCAA (Li et al., 2023), an interesting finding as chemotherapy may cause changes in gut microbiome (Wu et al., 2022).However, we did not measure gut microbiome in this study.
Our data on liver BCAA and LAT1 expression in both sexes indicates that the liver is the main receiver of muscle-derived BCAA.In the liver, AAs such as alanine and glutamine (Gannon & Nuttall, 2010), as well valine (Mann et al., 2021), which was found to be higher following drug treatment, can be converted into glucose by gluconeogenesis.Therefore, it is likely that the increase in liver BCAA is a compensatory mechanism in an attempt to not only remove excess BCAA from the plasma, but to also generate glucose at a time following chemotherapy when there are substrate deficits.
A limitation of this study is that glucose levels during our ITT could be confounded by chemotherapy-induced changes in baseline glucose.However, we observed no significant treatment effects on the slopes of the glucose curves during ITTs.Another limitation is that in real life, chemotherapy drugs are not given to healthy individuals.However, we have used clinically relevant chemotherapy drugs (Tabernero et al., 2015) to probe sex differences in metabolic responses, especially BCAA catabolism, to these drugs.The fact that elevations in liver BCAA concentrations (Ericksen et al., 2019;Zhang & Han, 2017) are seen in many cancers is consistent with observations made in this study, and suggests that the changes we observed will likely be heightened when tumor implantation is combined with chemotherapy, a subject that our lab is interested in.Lastly, we did not include interventional treatments here.However, since sex differences in BCAA metabolism following chemotherapy are rarely studied, it was difficult to decide on appropriate interventions.
Findings from this study offer possibilities for designing future interventional studies.
In conclusion, compared to male animals, drugtreated female animals experienced worsened outcomes for body and gastrocnemius muscle weights, and p-S6K1 thr389 signaling.On the other hand, drug-treated male animals were more insulin resistant and experienced more severe decreases in skeletal muscle BCAA concentrations.We did not directly study whether the altered BCAA metabolism has a causative effect.However, the positive correlations that we observed between both LAT1 and BCKD activity and muscle weight suggest that disruption of BCAA metabolism may play a role in cachexia and that interventions that can correct the disruption may help in the management of this condition.

F
I G U R E 6 The BCAA are positively correlated with gastrocnemius muscle weight, LAT1 transporter expression and BCKD activity.Correlations between the BCAA and gastrocnemius muscle weight (a), skeletal muscle LAT1 (BCAA transporter) expression (b) and skeletal muscle BCKD activity (c).Correlations between gastrocnemius muscle weight and LAT1 (d) and BCKD activity (e) are also shown.Data were analyzed using linear regression and 95% confidence intervals are denoted.These charts are drawn from the data in Figures 1, 3 and 4.
Summary of main findings of drug treatment and sex.